Smallpox has, historically, been one of the great killers of mankind. Although this disease is considered to have been eradicated some 35 years ago, the poxviruses, nonetheless, comprise a major family of viruses of medical, ecological and agricultural importance. For humans, aside from the possibility of smallpox re-introduction, eradication has coincided with the appearance of human monkeypox in Africa during the past 40 years and in the US during the past decade. Not knowing the lethal factor in smallpox, the full potential of such outbreaks remains uncertain. The importance of virus envelopes and capsid proteins in mediating the effects of antiviral therapeutics and vaccines is undisputed. From the picornaviruses to the herpesviruses, an understanding of virion outer molecular structures at molecular or atomic resolution, has instructed the rational design of therapeutic agents and an understanding of mechanisms that cause and thwart virus infection and disease. Due to their complexity, asymmetry and heterogeneity, the poxviruses have, however, been particularly persistent in defeating attempts to understand their virion structure at the molecular level, thus evading an important potential avenue for intervention. The P.I. hypothesizes that the complexity of the vaccinia virion may prove to be an Achilles heel. In addition, a full understanding of virion structure may be regarded as one of the last remaining black boxes in the lifecycle of the poxviruses - one which impinges upon the early transcription, genome uncoating and virion assembly stages of poxvirus replication. The major hole in our knowledge of pox virion structure lies at a level between the inventory of proteins present within the virion (which is largely known) and the basic topological and topographical features of the intact particle (also known). This intervening area may be referred to as the virion's molecular architecture, or protein interactome. In this R21 proposal, the P.I. has chosen a protein-protein crosslinking approach in combination with mass spectrometry. Such an approach for interactome analysis is unbiased in many respects, and has a track record of informing the molecular architectures of elaborate cellular assemblies such as the nuclear pore complex, 20S proteasome and RNA polymerase II. Aim 1 of this proposal seeks to identify directly juxtaposed proteins within the virion core via covalent protein-protein crosslinking/MS, taking top-down (protein-level) and bottom-up (peptide-level) approaches. The P.I. hypothesizes that the pox virion core wall may not be fundamentally dissimilar to the matrix protein layers of some enveloped RNA viruses, and that the classical delineation of enzymes in the deep interior with structural proteins surrounding may not be as clear cut as currently supposed.